【作者】 赵炜疆；

【导师】 李桂林； 袁芳；

【作者基本信息】 首都医科大学 ， 神经生物学， 2007， 博士

【摘要】 第一部分己烯雌酚诱发大鼠泌乳素腺瘤生成的形态学动态观察及血管生成相关因子表达研究背景与目的形态学改变是垂体瘤发生、发展和演进过程中最基本的表现之一,但有关垂体瘤发生的形态学的动态改变过程,具体研究较少。血管生成是多数肿瘤增大和浸润生长的结构基础和重要标志之一。本研究以实验性大鼠垂体泌乳素腺瘤模型为基础,结合影像学和组织形态学检查,从活体和组织学两方面对己烯雌酚诱导大鼠泌乳素腺瘤形成过程中腺垂体结构和血管生成相关因子表达的动态改变情况进行研究。材料和方法共用51只雌性Wistar大鼠,实验分溶剂对照组和模型组(DES组)。DES组大鼠接受腹腔注射己烯雌酚(5mg/kg,每周2次);相应给予溶剂对照组大鼠腹腔注射等体积葵花油,给药过程中监测大鼠体重和皮毛变化情况。分别于实验4w,8w和12w经心脏灌注固定,并取垂体组织进行形态学观察。使用HE染色进行肿瘤鉴定,并对不同时间点大鼠腺垂体的组织形态学改变进行评分。使用免疫组织化学观察肿瘤组织中泌乳素(PRL),促肾上腺皮质激素(ACTH),生长激素(GH)和黄体生成素(LH)的反应性,以确定肿瘤类型,同时检测大鼠垂体组织表皮生长因子(EGF)、血管内皮生长因子(VEGF)和血小板内皮细胞粘附分子(CD31)的反应性。麻醉灌注前于各组随机抽取大鼠3只进行磁共振成象(MRI)检查,观察活体条件下大鼠垂体组织的生长情况。结果1体重及一般表现随实验进展,溶剂对照组大鼠体重迅速增加,实验4w,8w和12w,DES组大鼠体重均明显低于对照组(均为p<0.01)。DES组大鼠自实验2周起表现为明显的脱毛,以腹壁及两侧以及枕后部为著。2 MRI扫描MRI动态观察显示,实验各时间点DES组大鼠矢状切面积均明显大于溶剂对照组(均为p <0.05),MRI显示垂体硬脑膜面明显膨隆,失去其固有的锐利外观。其底面无明显变化,表明肿瘤体积增大以向正上、前上及后上为主。3组织学观察及瘤细胞鉴定随着实验进展,接受DES的大鼠垂体开始出现增生、管腔变窄消失,既而大部分垂体结构被迅速生长的瘤样细胞占据;此后实体性瘤样结构中部分细胞胞核固缩。而同一组织切片中可观察到实体组织中有血管内皮细胞出现及管腔形成。各时间点肿瘤诱发率分别为40% (4/10)、60% (6/10)和80% (8/10)。免疫组化显示肿瘤为泌乳素型,间有ACTH阳性细胞散在分布,而生长素(GH)、黄体生成素(LH)表达阴性。4血管生成相关因子表达免疫组织化学显示在瘤样改变的垂体组织中可见到EGF、VEGF和CD31不同程度的表达。结论(1)DES诱发大鼠泌乳素腺瘤发生发展过程中,肿瘤主要以向上生长为主。组织学上,肿瘤发展是一个从增生到瘤变,从局部到整体,从实体性变化到新生血管融汇的一个动态发展和演进的过程,提示实验性垂体瘤发生进展过程中垂体自身形态转化具有重要意义,可能是垂体瘤由良转恶,由非侵袭性向侵袭性转化的结构基础。(2)体重降低及大鼠脱毛可作为己烯雌酚诱发大鼠泌乳素腺瘤发生的外在参考。(3)生长因子如EGF、VEGF和CD31等不同程度地表达于新生血管内皮细胞及/或瘤样细胞,表明它们在垂体瘤血管生成过程中发挥作用,并存在相互配合、协同作用的可能。第二部分非组织特异性中性半胱氨酸蛋白酶μ-及m-calpains在实验性泌乳素腺瘤发生中的表达及活性变化背景与目的垂体瘤约占颅内肿瘤15%,普通人群中垂体微腺瘤的临床检出率达10-25%。研究表明垂体瘤的形成、发展是垂体外因素和垂体内部因素多方面协同作用的结果。Calpains是依赖Ca2+的中性半胱氨酸蛋白酶家族,有着广泛的生物学作用。已有研究表明,calpain参与了多种内分泌细胞的分泌过程,然而calpains在垂体瘤发生发展过程的具体作用,有待深入探讨。本研究使用定期腹腔注射己稀雌酚(DES)诱导雌性Wistar大鼠泌乳素腺瘤形成,重点观察肿瘤形成过程中垂体组织calpains表达、活性变化。材料和方法93只雌性Wistar大鼠随机分入溶剂对照组和模型组(DES组),分别用于形态学和生化分子生物学研究。DES组大鼠接受腹腔注射DES(5mg/kg,每周2次);相应给予溶剂对照组大鼠腹腔注射等体积葵花油。于实验4w,8w和12w经心脏灌注固定部分大鼠,并取垂体组织进行形态学观察。使用免疫组织化学观察肿瘤组织中泌乳素(PRL),促肾上腺皮质激素(ACTH)的表达、分布,同时检测大鼠垂体组织中非组织特异性calpains(μ-和m-calpains)以及活性胱冬肽酶-3(caspase-3)的表达与分布。使用免疫荧光双标法观察两种calpains与泌乳素(PRL)的共位情况。其余大鼠用于生化分子生物学检查。于实验4w,8w和12w麻醉大鼠,腹主动脉采血3ml后,立即断头处死,取垂体组织,液氮冷却,-80℃保存备用。使用酶联免疫分析(ELISA)观察不同时间点血清中ACTH水平变化情况;使用蛋白质印迹(Western Blotting)法检测垂体组织胞浆中μ-和m-calpains表达,采用酪蛋白酶谱法观察不同时间点垂体组织胞浆及膜成分中μ-和m-calpains的活性变化。结果1诱发率各时间点肿瘤诱发率分别为40% (4/10)、60% (6/10)和80% (8/10)。2免疫组织化学及免疫荧光免疫组化显示肿瘤为泌乳素型,间有ACTH阳性细胞散在分布。与溶剂对照组相比,垂体组织中两种calpains表达均增强,而活性caspase-3仅选择性弱表达于垂体组织胞浆中。免疫荧光双标法显示m-calpain和μ-calpain与泌乳素共位于同一细胞中。3蛋白质印迹实验4w时间点,与溶剂对照组相比胞浆m-calpain表达明显升高,至8w时间点时显著下降,实验12w时又达到一定水平。而在DES组实验4w和8w时间点μ-calpain胞浆表达始终处于较低水平,至实验12w时则显著升高。4酪蛋白酶谱酪蛋白酶谱法显示实验各时间点,溶剂对照组大鼠垂体组织胞浆中均有一定水平的μ- calpain活性,而4w时DES组大鼠垂体组织胞浆μ-calpain活性为0,8w时开始出现,至12w时间点时明显升高。4w时两组膜成分μ-calpain活性均为0,而8w时溶剂对照组膜成分中开始出现μ- calpain活性,DES组仍为0,至12w时间点,溶剂对照组膜成分无明显μ- calpain活性,而在DES组则活性显著升高。4w时间点,m-calpain胞浆活性在DES组显著升高,至8w时下降,12w时又有一定程度的升高;4w及8w时间点,DES组膜成分中m-calpain活性仅维持较低水平,至12w时,该组膜成分中m-calpain活性显著升高。5 ELISA检测血清水平4w时间点,DES组血清ACTH水平显著下降,8w和12w时则ACTH血清水平与溶剂对照组相当。结论(1)采用腹腔注射己烯雌酚的方法可诱导大鼠泌乳素腺瘤,延长DES注射时间可增加肿瘤诱发率。(2)DES在诱导大鼠垂体泌乳素腺瘤形成过程的同时,促进了垂体组织即前叶(AL)和中间叶(IL)中两种Calpains的表达,其分布与泌乳素分布具有高度一致性,表明两者可能与泌乳素细胞的功能活动有关,是垂体泌乳素腺瘤病理生理表现的物质基础之一。垂体组织一定程度上天然缺乏活性caspase-3的表达,确保过度激活的calpains不因为激活caspase-3而引起细胞凋亡并造成分泌细胞损伤。(3)ACTH在IL的表达增加提示在DES作用下,中间叶细胞处于高度活跃状态,其可能通过旁分泌方式作用于垂体前叶,促进泌乳素分泌,从而在泌乳素腺瘤形成过程中发挥作用。(4)深入研究Calpains在激素分泌调控中及其在诱瘤过程中垂体各部位相互关系中的作用,有助于从分泌等环节抑制激素分泌,改善临床症状。第三部分褪黑素(melatonin)在实验性大鼠泌乳素腺瘤治疗中的机制研究背景与目的:泌乳素腺瘤是临床上常见的垂体瘤类型,实验性大鼠泌乳素腺瘤以血清泌乳素水平升高和血管增生为主要表现。褪黑素(melatonin)在各种实验诱导的内源性肿瘤生成以及离体条件下动物和人的细胞系中都具有明显的抗肿瘤作用,但其抗肿瘤生长作用机制并不十分清楚。本研究观察褪黑素治疗DES诱导大鼠泌乳素腺瘤过程中血管内皮生长因子(VEGF)、表皮生长因子(EGF)、血小板内皮细胞粘附分子(CD31)和两种非组织特异性calpains的表达、活性的变化情况,进而探讨褪黑素治疗垂体泌乳素腺瘤的可能机制。材料和方法:64只雌性Wistar大鼠,实验分5组:组1:腹腔注射葵花油(1ml/kg,每周两次)共计16w;组2:腹腔注射己烯雌酚(DES)(5mg/kg,每周两次),共计16w;组3:腹腔注射DES(5mg/kg,每周两次),连续12w,之后停止给予DES至第16w结束;组4:腹腔注射DES(5mg/kg,每周两次),于实验第13周开始在继续行己烯雌酚注射的同时给予给予皮下注射褪黑素(0.25mg/d),至第16w结束;组5:腹腔注射己烯雌酚溶液(1mg/kg,每周两次),连续12w,并于实验第13周开始在继续行己烯雌酚注射的同时给予给予皮下注射褪黑素(0.25mg/只/天和1.0mg/只/天),至第16w结束。给药前称重,并观察各组大鼠毛发变化情况。实验结束时,灌注部分大鼠,取垂体组织,石蜡包埋,分别用于HE染色,以及免疫组织化学观察EGF、VEGF和CD31的表达变化。另有部分大鼠腹主动脉采血3ml后,立即断头处死,取垂体组织,液氮冷却,-80℃保存备用。使用酶联免疫分析(ELISA)观察不同组血清中ACTH水平变化情况。使用蛋白质印迹(Western Blotting)法检测垂体组织胞浆中μ-和m-calpains表达,采用酪蛋白酶谱法观察不同时间点垂体组织胞浆及膜成分中μ-和m-calpains的活性变化。结果:1体重及一般表现长期注射DES使大鼠体重明显降低,毛发脱落,而停止给予雌激素或给予不同剂量褪黑素后,则大鼠体重增加,毛发状况改善。2 HE评分长期接受DES后,大鼠垂体组织学评分明显增加,而停止给予DES或给予不同剂量褪黑素后则其组织学评分下降。3 EGF、VEGF和CD31表达变化长期接受DES后,大鼠垂体组织EGF、VEGF以及CD31的表达显著增强,而停止给予DES则三种因子表达下降,并可被不同剂量褪黑素抑制。4电镜观察瘤样改变区细胞出现多形性分泌颗粒、片层小体和错位性胞溢,停止给予DES或给予不同剂量褪黑素后,错位性胞溢消失,分泌颗粒外排受阻、粗面内质网和高尔基体向正常结构转化。5μ-和m-calpain胞浆表达及酪蛋白水解活性长期给予DES垂体组织胞浆μ-和m-calpain水平升高,膜成分活性增高,而停止给予DES或使用不同剂量褪黑素可不同程度降低胞浆μ-和m-calpain水平,抑制两种非组织特异性calpains的激活。6血清ACTH水平长期给予DES,血清ACTH水平略有下降,停止给予DES或给予不同剂量褪黑素后,则血清ACTH水平有所升高。结论(1)长期腹腔注射DES可诱导大鼠泌乳素腺瘤形成,有较稳定的诱发率,血管形成和和瘤细胞增生比较明确,是观察实验性大鼠泌乳素腺瘤治疗的较为理想的模型。相关指标如大鼠体重、毛发状况可作为大鼠对DES敏感性及药物治疗的外在参考。血清ACTH水平可作为褪黑素疗效和调整治疗剂量等的一个参考。(2)在大鼠泌乳素腺瘤形成的过程中,垂体组织中EGF、VEGF和CD31的表达增高,并具有一定的雌激素依赖性。不同剂量的褪黑素可减少上述因子在垂体组织细胞、血管内皮的表达,表明褪黑素可通过抑制血管生成相关因子来抑制垂体瘤发生。(3)在大鼠泌乳素腺瘤形成的过程中,垂体组织胞浆中calpains表达增高,并具有一定的雌激素依赖性,可能与DES诱发大鼠泌乳素腺瘤过程中高分泌和血管生成维持有关。不同剂量的褪黑素可减少calpains的胞浆表达,从而抑制其对高分泌和血管生成的维持。(4)长期给予DES后,垂体组织中calpains的激活明显增加,并具有一定的雌激素依赖性。褪黑素可通过稳定胞浆中calpains,抑制其转膜激活,对抗DES对大鼠垂体组织的影响。褪黑素抑制泌乳素颗粒释放,可能与其抑制calpain激活,囊泡脱包被和成熟受阻有关。(5)血管生成相关因子高表达、calpain表达增高和过度激活在实验性垂体泌乳素腺瘤的发生发展过程中具有一定的协同作用,褪黑素可能是通过单独或在信号转导水平作用于上述靶点,发挥作用。两种calpains在实验性泌乳素腺瘤形成中的具体分工仍有待进一步明确。更多还原

【Abstract】 Part 1 Study on dynamic morphological changes and expressions of angiogenesis-related factors during the development of diethylstilbestrol (DES)-induced prolactinoma in ratsBackground and purpose Morphological changes are one of the basic signs of the initiation, development and formation of pituitary tumor. However, the dynamic morphological changes of pituitary tissues during tumor development are rarely discussed. Angiogenesis is the constitutive base and vital mark of tumor enlargement and invasion. Based on experimental pituitary tumor induced by diethylstilbestrol (DES) in rats, we explored the dynamic morphological changes of pituitary tissues and expressions of angiogenesis-related factors during the formation of DES-induced pituitary tumor.Methods 51 female Wistar rats were randomly allocated into vehicle-controlled group and DES group. Rats in DES group received intraperitoneal injection of DES (5mg/kg, twice a week), while their controls were correspondingly administered with sun-flower seed oil. At 4 week (w), 8w and 12w, animals were intracardially perfused and pituitary tissues were harvested for morphological investigations. HE staining was used to confirm the occurance of tumor, and each section was scored based on HE observations. Immunohistochemical method was undertaken to identify the tumor phenotype, in which PRL, ACTH, GH, and LH reactivities were examined. Expressions of three angiogenesis-related factors: epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) and CD31 were investigated. Body weight and fur change were observed at each time point. Prior to perfusion, MRI scans were undertaken in 3 randomly selected rats from each group to study the in vivo growth profile of the pituitary gland.Results 1 Body weight and appearance changes A rapid body weight gaining was observed in vehicle-controlled rats, whose body weights were 189.6±15.6g, 256.4±27.0g, and 244.5±23.8g after 4, 8 and 12 weeks of vehicle treatment. In contrast, rats in DES-treated group were 164.0±11.4g, 209.2±13.5g and 208.4±21.0g, significantly decreased compared with their controls (p<0.01 at each time point). Rats in DES-treated group began losing hair at 2 week, which appeared more severely in costal region and backneck。MRI The mid-sagittal areas of DES-treated pituitary were significantly larger compared to their controls at each time point (p<0.01 at each time point) based on T2WI investigations. MRI scans also demonstrated gradually upward enlargement of the pituitary tissue with no apparent changes in its base. Histological observations and tumor phenotype identification Hyperproliferation and gland cavity narrowing initially occurred in DES-treated pituitary tissues, which were then rapidly occupied by tumor-like cells. Afterwards, nuclear condensation was observed within areas with tumor-like changes. Endothelial cell emergence, double-wall emergence and cavity extension were concurrently observed in the same section. At each time point, the incidence rates of tumor were 40% (4/10), 60% (6/10) and 80% (8/10). Immunohistochemical stainings confirmed the tumor phenotype as prolactinoma, with dotted distribution of ACTH positive cells within these areas. Expressions of GH and LH were negative. Expressions of angiogenesis-related factors Each stage of the development of angiogenesis saw expressions of EGF, VEGF, and CD31 in the endothelial cells, and /or vascular walls and peripheral cells in areas with tumor-like changes.Conclusions (1) The growth of the pituitary gland is characterized by upward extension during the development of pituitary tumor. (2) Histologically, the thorough process of tumor development was composed of hyperproliferation to tumorigenesis, local to whole, solid to mixture joined by newly-generated vessels. The self reconstruction of pituitary tissues represents a morphological base for the transformation of pituitary tumor from benign to malignant, and from non-invasion to invasion. (3) Body weight reduction and fur loss can be regarded as signs of the development of pituitary tumor under the induction of DES. (4) EGF, VEGF, and CD31 were expressed in the endothelial cells and pituitary cells in their respective manner, suggesting they may function and cooperate with each other at each stage of angiogenesis.Expressions and activities of two ubiquitous calpains (μ-and m-calpains) during the development of diethylstilbestrol (DES)-induced prolactinoma in ratsBackground and Purpose Frequently occurred, the pituitary tumors account for 15% of the intracranial tumors, with high frequency of the pituitary microadenoma among normal people. Studies have demonstrated that the development and formation are based on the mutual actions between intrapituitary and extrapituitary factors in different levels. Calpains represent a Ca2+ dependent protease family comprised of more than 14 members, among whichμ- and m-calpains are ubiquitous calpains widely distributed in a variety of tissues, where they exert extensive biological functions. Data available have demonstrated the involvement of calpains in the secretion process of several lines of endocrine cells. However, roles of calpains in the development of pituitary tumor are rarely discussed. Based on the rat model of diethylstilbestrol (DES)-induced prolactinoma, we focused our main research on the expressions, activities of calpains and their possible relationships to prolactin secretion during the development of prolactinoma.Material and Methods 93 female Wistar rats were randomly allocated into vehicle-controlled group and DES-treated group, which were used for morphological and biomolecular investigations. In DES group, rats received DES intraperitoneally (5mg/kg, twice a week), and in the vehicle-controlled group, rats were intraperitoneally injected sun-flower seed oil as vehicle (1ml/kg, twice a week). At 4 week (w), 8w and 12w, some animals were intracardially perfused and pituitary tissues were harvested for morphological investigations. HE staining was used to confirm the occurance of tumor. Immunohistochemical method was employed to study the expressions of prolactin (PRL) and ACTH in areas with tumor-like changes. Expressions and distributions of two ubiquitous calpains as well as their downstream effector caspase-3 were also visualized by immunohistochemial investigation. Double immunofluorescence-labeling were used to cofirm the potential relationship betweenμ-calpain and PRL, and that between PRL and m-calpain. At the same time point, some other animals were anaesthetized, and the abdomen aorta was exposed for blood collection, soon after which the pituitary tissues were harvested for future studies after fluid N2 freezing. ELISA was used to study the serum level of ACTH. Cytosol expressions of calpains at each time point were investigated by using Western Blotting. Zymography was undertaken to study the cytosol and membrane activities of both ubiquitous calpains.Results 1 induction rate The induction rates in DES-treated group at each time point were 40% (4/10), 60% (6/10) and 80%(8/10). 2 immunohistochemistry and immunofluorescence Pituitary tissues with tumor development demonstrated a strong reactivity to prolactin and dotted ACTH positivity. In tumor located regions, expressions of bothμand m-calpain were increased, although physiological expressions of two ubiquitous calpains were seen in both the anterior lobe and the intermediate lobe. Double immunofluorescence-labeling confirmed the co-localization ofμ-calpain and PRL, as well as that of m-calpain and PRL. The distributions of two calpains were similar to that of PRL, whose secretion is uniquely characterized by extravasation in prolactinoma. 3 Western Blotting At 4w, cytosol expression of m-calpain was significantly increased, followed by a sharp reduction at 8w and an apparent increase at 12w. However, cytosol expressions ofμ-capain in DES group remained low levels at both 4w and 8w, followed by a significant increase at 12w. 4 Casein Zymography (ⅰ) Low-leveled cytosol activities ofμ-calpain were seen in the veicle-controlled group at each time point. In contrast, cytosol activity ofμ-calpain in DES group was 0 at 4w, followed by its emergence at 8w and significant increase at 12w. (ⅱ) The membrane activity ofμ-calpain in vehicle-controlled was present only at 8w. Its activity remained 0 at both 4w and 8w in both DES groups, and was significantly increased at 12 week. (ⅲ) At 4w, a comparatively high-leveled cytosol m-calpain activity was present in the DES group, followed by a sharp reduction at 8w, and an apparent reincreasing at 12w. (ⅳ) The membrane m-calpain activities remained low levels in the DES group at both 4w and 8w, followed by a significant increase at 12w. 5 ELISA for serum ACTH At 4w, the serum ACTH level was significantly reduced, and was restored to levels similar to those in the vehicle-treated group at 8w and 12w.Conlusions (1) Intraperitoneal injection of DES can induce prolactinoma in rats, and prolonged injection increased the incidence rates of prolactinoma. (2) With the induction of the development of prolactinoma, DES promoted the expressions of two ubiquitous calpains in both AL and IL. The two calpains were colocalized with PRL intracellarly in the AL, suggesting they are highly related to the functions of prolactatrophs and may represent one of the substantial bases of the pathophysiological functions of pituitary tumor. Cleaved caspase-3 was selectively and weakly expressed in hypophyseal cells, excluding the possibility of its activation by highly activated calpains, leading to apoptosis-induced cellular injury. (3) The subsequent restoration of serum ACTH occurred, in concurrence with the enhanced immunoreactivity of ACTH in the IL, suggesting the formation of hyperactivity in this hypophyseal part, which may exert roles in prolactin expressin and secretion in the AL through secreting several paracrine factors. (4) To explore the roles of two ubiquitous calpains in hormone secretion may be of vital importance in prolactinoma treatment clinically.Mechanisms for the treatment of melatonin in DES-induced prolactinoma in female Wistar rats Background and Purpose Prolactinoma is a type of frequently-occurred pituitary tumor clinically, which is mainly characterized by high serum prolactin and angiogenesis. In vivo and in vitro studies have demonstrated the anti-tumor effect of melatonin, but the detailed mechanisms remain elucidation. In the present research, we studied the effects of melatonin on expressions of angiogenesis-related factors EGF, VEGF, and CD31, as well as the expressions of bothμ-and m-calpains and their Ca2+-dependent activities during the development of DES-induced prolactinoma in female Wistar rats.Materials and Methods 64 female Wistar rats were randomly allocated into 5 groups. In group 1, rats received intraperitoneal injection of sun flower seed oil for 16 weeks. In group 2, rats were intraperitoneally administered with DES (5mg/kg, twice a week) for 16 weeks, while rats in group 3 was administered with DES for 12 weeks, followed by its discontinuation for 4 weeks. In group 4 and 5, rats received DES for 16 weeks, with subcutaneous administration of melatonin at doses of 0.25mg/day and 1.0mg/day at the beginning of the time point of 13 week. Body weight and fur changes were observed before each injection. Some rats were subjected to perfusion to harvest the pituitary tissues, which were used for HE staining, immunohistochemical studies for expressions of EGF, VEGF and CD31. In the other rats, 3ml of blood were collected from the abdomen aorta, which was used for determining the serum ACTH level by using ELISA. The pituitary tissues were harvested for cytosol expressions of two ubiquitous calpains and their cytosol and membrane activities.Results 1 Body weitht and fur Long-term treatment of DES significantly reduced body weight and fur loss, which could be inhibited by DES withdrawal and melatonin administration at different doses. 2 HE score Long-term treatment of DES significantly increased the HE score, which could be reduced by DES withdrawal and melatonin at different doses. 3 Expressions of EGF, VEGF and CD31 Expressions of EGF, VEGF, and CD31 were greatly enhanced after long-term DES administration, which could be reduced by DES withdrawal and melatonin at different doses. 4 Electronic microscopy Tumor-like cells contained multi-shaped secretion granuales, sheet-like bodies of the rough endoreticulum (RER) and displayed misplaced exocytosis. After DES withdrawal and melatonin treatment, misplaced exocytosis disappeared, granule extrusion was arrested. 5 Cytosol expressions of two ubiquitous calpains and their in vitro activities The cytosol expressions of two ubiquitous calpains were increased due to continuous treatment of DES for 16 weeks, with their membrane activities increased. DES discontinuation or melatonin administration at 2 doses could significantly reduce their cytosol expressions and in vitro activities. 6 Serum ACTH level A slight reduction of serum ACTH level was observed in group 2, and DES discontinuation and melatonin treatment could restore its serum level in a certain degree.Conclusions 1 Long term intraperitoneal administration of DES could induce angiogenesis and hyperproliferation in the pituitary gland in female Wistar rats, which provides an idea model for experimental prolactinoma treatment. Body weight and fur changes could be used to evaluate reaction of rats to DES and medical treatment. Serum ACTH level could be referred for modulating drug dose. 2 Expressions of EGF,VEGF, and CD31 were DES dependent, and melatonin may exert its anti-tumor effect by downregulating expressions of EGF, VEGF and CD31. 3 Increased cytosol expressions and upregulated membrane activities of bothμ-and m-calpains may be involved in the development of prolactinoma. And melatonin may reduce granule extrusion by inhibiting activities of two ubiquitous calpains. 4 The concurrence of high expressions of angiogenesis-related factors and cytosol calpains as well as their increased membrane activities indicated their cooperation during the development of experimental prolactinoma, which may represent the treatment targets of melatonin. The detailed roles of two ubiquitous calpains need further investigations.更多还原